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Mol Biol Cell. 2014 Nov 5;25(22):3610-8. doi: 10.1091/mbc.E14-06-1091. Epub 2014 Sep 17.

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Author information

1
European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
2
European Molecular Biology Laboratory, 69117 Heidelberg, Germany ellenberg@embl.de.

Abstract

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

PMID:
25232003
PMCID:
PMC4230620
DOI:
10.1091/mbc.E14-06-1091
[Indexed for MEDLINE]
Free PMC Article

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